Breaking down miRNAs

Although much work has examined microRNA (miRNA) biogenesis, relatively little is known about miRNA decay. Elbarbary et al. now identify Tudor-SN, an endonuclease that interacts with the RNA-induced silencing complex. Tudor-SN targets miRNAs at CA and UA dinucleotides located more than five nucleotides from miRNA ends. Tudor-SN-mediated miRNA decay removes miRNAs that silence genes encoding proteins that are critical for the G1-to-S phase transition in the cell cycle.

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. The pathways that mediate mature miRNA decay are less well understood than those that mediate miRNA biogenesis. We found that functional miRNAs are degraded in human cells by the endonuclease Tudor-SN (TSN). In vitro, recombinant TSN initiated the decay of both protein-free and Argonaute 2–loaded miRNAs via endonucleolytic cleavage at CA and UA dinucleotides, preferentially at scissile bonds located more than five nucleotides away from miRNA ends. Cellular targets of TSN-mediated decay defined using microRNA sequencing followed this rule. Inhibiting TSN-mediated miRNA decay by CRISPR-Cas9 knockout of TSN inhibited cell cycle progression by up-regulating a cohort of miRNAs that down-regulates mRNAs that encode proteins critical for the G1-to-S phase transition. Our study indicates that targeting TSN nuclease activity could inhibit pathological cell proliferation.

We first confirmed that cellular TSN coimmunoprecipitates with the RISC components AGO2 and trinucleotide repeat–containing 6A (also known as GW182) in a largely RNase I (ribonuclease I)–resistant fashion (fig. S1A and table S1). We corroborated this result by detecting cellular TSN in the reciprocal immunoprecipitation of FLAG-tagged AGO2 (fig. S1B).

To determine whether RISC-associated TSN functions in miRNA metabolism, we quantitated the levels of select miRNAs and their precursor miRNAs (pre-miRNAs) and pri-miRNAs in human embryonic kidney (HEK) 293T cells transfected with TSN small interfering RNA (siRNA) relative to control (Ctl) siRNA (fig. S2A). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Northern blotting demonstrated that TSN knockdown up-regulates the abundance of mature miR-31-5p, miR-29b-3p, and miR-125a-5p without significantly changing the levels of their pre- or pri-miRNAs (Fig. 1A and fig. S2B). TSN knockdown did not alter the abundance of mature miR-3648 and miR-128-3p (fig. S2C), which suggests that these effects are not generalizable to all miRNAs. These same miRNA-specific results were observed with HeLa cells (fig. S2D).

(A) RT-qPCR revealing that mature miRNA levels increased in cells transfected with TSN siRNA relative to Ctl siRNA, while the corresponding pre- or pri-miRNA levels remained unchanged. (B) Western blot showing that exposing Dicer-kd 2b2 cells (20) to doxycycline (Dox) to induce Dicer short hairpin RNA (shRNA) production inhibits Dicer expression. Cells were transfected with Ctl or TSN siRNA and exposed to Dox for 4 days before harvesting. (C) RT-qPCR of RNA from (B) demonstrating that when doxycycline is used to inhibit miRNA biogenesis, miRNAs are stabilized by TSN siRNA relative to Ctl siRNA. (D) RT-qPCR showing that TSN siRNA up-regulates the level of exogenously introduced miR-31-5p relative to the level of co-introduced miR-3648. (E) RT-qPCR showing that wild-type TSNR, but not the catalytically inactive variant TSNR(ΔSN1) or TSNR(ΔSN4), rescues the activity of cellular TSN. Here and elsewhere, all results derive from three or more independent experiments. For RT-qPCR results, miRNA and pre-miRNA levels are relative to U6 snRNA, pri-miRNA levels are relative to β-actin mRNA, and relative levels in the presence of Ctl siRNA are defined as 100. Histograms represent the average and SD. *P < 0.05, **P < 0.01.

TSN targets functional miRNAs, as evidenced by the finding that TSN knockdown and the consequential accumulation of miR-31-5p and miR-29b-3p (Fig. 1A) are accompanied by decreased expression of integrin α5 (ITGA5) and lysyl oxidase (LOX) (fig. S3A), which are encoded by their respective mRNA targets (6, 7) (fig. S3, B to E). The decreased level of ITGA5 and LOX was restored by expressing a miR-31-5p inhibitor or a miR-29b-3p inhibitor, respectively (fig. S3A); this result confirmed that the observed TSN regulation is miRNA-mediated.

The increased abundance of miRNAs and their mRNA-targeting activities upon TSN knockdown is not due to enhanced loading of miRNAs into AGO2. This was made clear by programming FLAG-AGO2 in lysates of HEK293T cells, which stably express FLAG-AGO2 and had been transiently transfected with Ctl or TSN siRNA (fig. S3F), with 5′-[32P]-miR-31-5p (fig. S3G). Immunoprecipitations of FLAG-AGO2 revealed that TSN knockdown did not increase the level of 5′-[32P]-miR-31-5p in FLAG-AGO2 (fig. S1C).

Additional support for TSN promoting miRNA turnover derives from our finding that TSN knockdown increased the abundance of miR-31-5p, miR-29b-3p, and miR-125a-5p when miRNA biogenesis was inhibited by conditional Dicer knockdown (Fig. 1, B and C, and fig. S4, A to C), but showed no effect on the corresponding miR* (i.e., the less abundant strand of the Dicer-generated duplex) (fig. S4D). Furthermore, TSN knockdown increased the abundance of miR-31-5p that derived from an exogenously introduced miR-31-5p mimic (Fig. 1D), indicating that TSN-mediated miRNA turnover does not require Drosha- or Dicer-dependent processing steps that precede miR:miR* duplex formation. We term this TumiD (i.e., Tudor SN–mediated miRNA decay). TumiD adds a new dimension to reports that miRNA stability can be regulated by exonucleases and complementarity to target mRNAs (8).

Two independent microRNA sequencing (miR-seq) experiments—runs 1 and 2, each in biological triplicate—expanded the number of cellular TumiD targets. Upon TSN knockdown, the levels of 49 and 88 miRNAs were significantly up-regulated (adjusted P value < 0.05) in runs 1 and 2, respectively, of which 35 were common to both experiments (Fig. 2A, figs. S5 to S10, and tables S2 to S4).

Nine of 10 chosen miRNAs that were significantly up-regulated in both runs (Fig. 2A and table S4) proved to be bona fide TumiD targets (Fig. 2B and fig. S11A). Evaluation of the tenth, miR-221-3p, was obscured by up-regulation of its pri-mRNA upon TSN knockdown (fig. S11B). Using miR-seq and RT-qPCR, we demonstrated that TSN knockdown did not alter the abundance of the miR* for miR-125a-5p, miR-99b-5p, or miR-98-5p (fig. S11C and table S3), thereby providing additional evidence that TumiD degrades mature miRNAs. Note that the miR* of some TumiD targets is itself a TumiD target, as exemplified by miR-126-5p (Fig. 2, A and B, and tables S3 and S4).

TumiD also contributes to the differential regulation of the six miR-17-92 cluster–derived miRNAs (11) (fig. S11, D and E, and table S3) and of the miR-15a/16-1 and miR-23a/27a/24-2 cluster-derived miRNAs (fig. S12, A to C, and table S3). Notably, miRNA members of each cluster have distinct functions in development and disease.

TSN preferentially cleaves at CA and UA dinucleotides, as revealed when 5′-[32P]-miR-31-5p, 5′-[32P]-miR-29b-3p, or 5′-[32P]-miR-126-3p was incubated with recombinant histidine-tagged TSN (HIS-TSN) and reaction products were analyzed in sequencing gels (Fig. 3A and fig. S13A). Endonucleolytic cleavage was followed by 3′-to-5′ exonucleolytic degradation (Fig. 3A and fig. S13A), as is typical for the mode of the bacterial homolog of TSN, micrococcal nuclease (1). miR-3648, which lacks CA and UA, was resistant to HIS-TSN–mediated degradation (fig. S13B), as was a hybrid miRNA generated by swapping a cleavable CA-containing region of miR-31-5p with a region from miR-3648 (fig. S13C, compare to Fig. 3A). HIS-TSN–mediated degradation depended on Ca2+ (fig. S13B) and was inhibited by converting the phosphodiester bond of a susceptible dinucleotide to a phosphorothioate bond (Fig. 3, B and C, and fig. S14), which has lower affinity for Ca2+ (9). Inserting a CA dinucleotide into HIS-TSN–resistant miR-3648 induced cleavage at the inserted CA (fig. S15), and cleavage was least optimal at CA dinucleotides situated within the five 5′-most or five 3′-most nucleotides of a miRNA (fig. S16, A and B). Consistent with this finding, when the 11 3′-most nucleotides of miR-29b-3p were swapped for the corresponding region of miR-150-3p (which contains a CA in the five 3′-most nucleotides), cleavage in the swapped region was abolished (fig. S16, C and D, compare to Fig. 3A).

(A) Phosphorimages of sequencing gels reveal that recombinant TSN cleaves protein-free 5′-[32P]-labeled miRNAs at CA and UA. Alk, alkaline hydrolysis to generate a ladder of nucleotides (nts); T1, RNase T1 digestion to generate a ladder of nucleotides cleaved after guanines; (–), no HIS-TSN. Black, gray, and white arrowheads respectively denote strong, medium, and weak cleavage sites. Unlabeled bands are decay intermediates resulting from TSN exonuclease activity since they were not detectable upon 3′-[32P]Cp-labeled miRNA incubation with HIS-TSN (fig. S13A). (B) Replacing a susceptible CA phosphodiester bond in protein-free 5′-[32P]-miR-29b-3p with a CA phosphorothioate bond inhibits HIS-TSN–mediated degradation. WT, wild type; 17PS18, phosphorothioate bond between nucleotides 17 and 18. (C) Plot of results shown in (B). The amount of each 5′-[32P]-labeled miRNA in the absence of HIS-TSN is defined as 100. (D) Protein-free 5′-[32P]-miR-31-5p (–) is degraded by HIS-TSN(WT), endonuclease RNase I, or 3′-to-5′ exonuclease Exo T but not catalytically inactive (fig. S4G) HIS-TSN(ΔSN4). (E) FLAG-AGO2–loaded 5′-[32P]-miR-31-5p is protected from degradation by Exo T and is susceptible to degradation by HIS-TSN(WT) and RNase I but not by HIS-TSN(ΔSN4).

Investigating cellular TumiD revealed that artificially inserting CA dinucleotides into TumiD-resistant miR-3648 at two positions favorable for TSN cleavage in vitro (fig. S17A) converted the miRNA to a TumiD target (fig. S17B). Of the 35 miRNAs that were significantly up-regulated upon TSN knockdown in both miR-seq runs (table S4), the two lacking CA or UA were up-regulated before miRNA generation (fig. S17C); the remaining 33 contained CA and/or UA at favorable positions for TSN cleavage. Notably, 31 mouse miRNAs that we identified as orthologs of putative or proven human TumiD targets that were up-regulated upon TSN knockdown in both runs are themselves predicted to be TumiD targets and have CA and/or UA dinucleotides at the same position(s) (table S5).

We are underestimating the number of cellular TumiD targets, because miRNAs with half-lives sufficiently long to preclude a detectable change during the period of TSN knockdown will escape definition. We limited our analyses to ≤4 days after transient transfection using TSN siRNA because longer transient knockdown or constitutive knockout (KO) of TSN inhibits cell cycle progression (see below). Moreover, unknown factors must affect the specificity of cellular TumiD, given that short-lived miR-503-5p (12) is not a TumiD target despite harboring an internal CA (fig. S17D).

AGO2-loaded miRNAs are protected from 3′-to-5′ exonucleolytic degradation by bacterial exonuclease T (Exo T). However, we found that they were susceptible to endonucleolytic degradation mediated by bacterial RNase I, which cleaves between any two nucleotides, or HIS-TSN (Fig. 3, D and E, and figs. S4G and S18).

To assess whether TSN facilitates the G1/S-phase transition in human cells as it does in mouse embryonic fibroblasts (13), we established two independent stable TSN KO HEK293T cell lines using CRISPR, the D10A nickase variant of Cas9 (14), and, to control for off-target effects of CRISPR-Cas9, different pairs of single guide RNAs (fig. S19). Synchronizing wild-type and TSN KO cells at G2/M using nocodazole, or at G1/S using a double-thymidine block followed by flow cytometry analysis after release from synchronization, revealed a prolonged cell cycle in both TSN KO cell lines (TSN KO 1 or 2) due to slow progression through the G1/S transition (Fig. 4A and fig. S20). Stably expressing FLAG-TSN in each TSN KO cell line rescued the pace of G1/S progression (fig. S21, A and B). Consistent with results from transient TSN knockdowns (Fig. 1A and fig. S11E), the levels of the TumiD targets miR-31-5p, miR-17-5p, and miR-20a-5p were significantly up-regulated in G1/S in both TSN KO cell lines relative to wild-type cells (Fig. 4B and fig. S22A).

(A) Flow cytometry analyses of the cell cycle demonstrating delayed G1-to-S phase progression in TSN KO 1 cells compared to WT cells. Cells were synchronized in G2/M using nocodazole, released from synchronization, and analyzed at the designated times. The percentage of cells in each cell cycle phase is shown. (B) RT-qPCR showing up-regulation of TumiD targets in TSN KO 1 cells relative to WT cells. Cells were synchronized at G1/S using a double-thymidine block. (C) RT-qPCR demonstrating that the levels of CDK2, CCND1, E2F1, and E2F2 mRNAs are down-regulated in TSN KO 1 cells relative to WT cells when synchronized using a double-thymidine block, and that transfecting TSN KO 1 cells with miRNA inhibitors rescues the levels of CDK2, CCND1, E2F1, and E2F2 mRNAs.

Using miRNA mimics, we confirmed previous reports (15–18) that these three TumiD targets down-regulate the expression of genes critical for G1/S transition: Cyclin-dependent kinase 2 (CDK2) mRNA was down-regulated by a miR-31-5p mimic, both cyclin D1 (CCND1) and E2F transcription factor 1 (E2F1) mRNAs were separately down-regulated by a miR-17-5p mimic and a miR-20a-5p mimic, and E2F2 mRNA was down-regulated by a miR-20a-5p mimic (fig. S22B). Consistent with these results, increased G1/S levels of the three miRNAs in TSN KO cells were accompanied by decreased levels of their target CDK2, CCND1, E2F1, and E2F2 mRNAs (Fig. 4C and fig. S22C) and target-encoded proteins (fig. S22D). Furthermore, transfecting TSN KO cells with miRNA inhibitors rescued the levels of CDK2, CCND1, E2F1, and E2F2 mRNAs (Fig. 4C and fig. S22C) and their corresponding proteins (fig. S22D), indicating that TSN regulates the expression of these cell cycle regulatory proteins via TumiD. Although the level of E2F3 mRNA was also down-regulated in TSN KO 1 cells, miRNA inhibitors did not restore the level of E2F3 mRNA (fig. S22E), indicating that its down-regulation by TSN is not mediated by miR-17-5p or miR-20a-5p. Stably expressing FLAG-TSN in TSN KO cells rescued the expression levels of the three miRNAs and their target CDK2, CCND1, E2F1, and E2F2 mRNAs (fig. S22, F and G).

Our studies describe an endonuclease-mediated miRNA decay pathway that degrades specific miRNAs. In addition to adding an important dimension to how cellular miRNAs are regulated, these properties of TSN may be useful as a biochemical tool to degrade specific small RNAs in vitro.

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